1. Field of the Invention
There is an ever increasing need to covalently attach small molecules (haptens, enzyme substrates or inhibitors, reporter groups, etc.) to larger molecules (carriers, enzymes, insoluble matrices, etc.) for a variety of applications (vaccines, antibody production, immunoassays, isolation and separation techniques, etc). In many instances the small molecule is a peptide, which can be synthesized by current methodology. However, the polyfunctional nature of both the peptides and the larger molecule (e.g., proteins) does not allow for precisely controlled covalent attachment. The most commonly used coupling reagents are for the most part bifunctional, with the two functions of equal activity, frequently being the same functionality. Conventional reagents include dialdehydes, carbodiimides, diimidates and diesters, which tend to cross-link the proteins intramolecularly and intermolecularly, resulting in a low efficiency of the desired peptide carrier linkage, as well as substantial modification of the carrier molecules.
Since activation in aqueous media is very nonproductive, the stoichiometry of the conjugation is usually quite poor, i.e., only a small portion of the peptide used becomes attached to the larger molecule. Moreover, it is frequently desirable to know which functional group in the small molecule was utilized for linkage to the larger molecule.
Thus, there is an important need to develop methods which allow a well designed coupling site for the smaller molecule and are stoichiometrically acceptable for the conservation of materials in limited supply.
2. Description of the Prior Art
U.S. Pat. No. 4,127,526 describes the preparation of oligopeptides on chloromethylated resins. 4-Hydroxy-3-nitrobenzenesulfonic acid salt esters are reported in Klausner et al., in Peptides Proceedings of the Fifth American Peptide Symposium, Goodman, M. and Meienhofer, J., eds. John Wiley & Sons, New York, pp. 536-538. The N-9-fluorenylmethyloxycarbonyl (FMOC) is described by Chang et al., Int. J. Peptide and Protein Res. (1980) 15:485-494.